Article Figures & Data

Figures

Decreased amyloid deposition in BRI-Aβ40/Tg2576 mice. A, B, Representative entorhinal/piriform cortex sections from Tg2576 and BRI-Aβ40/Tg2576 mice at 15 (A) and 20 (B) months of age were immunostained with 33.1.1 (anti-Aβ1-16; top panels) or stained with ThioS (bottom panels). Scale bars: (in A) top panels, 200 μm; bottom panels, 50 μm. C, D, The amyloid plaque burden and number of ThioS-positive cored plaques in the entorhinal/piriform cortex (Ctx) and hippocampus (Hip) at 15 (C) and 20 (D) months were quantified. There was a significant decrease in both the Aβ plaque burden and number of ThioS-positive plaques in BRI-Aβ40/Tg2576 mice compared with Tg2576 littermates (p < 0.05). For statistical analysis, see Materials and Methods.

Decreased Aβ accumulation in BRI-Aβ40/Tg2576 mice. A, RIPA-insoluble, FA-extractable Aβ40 and Aβ42 levels in the forebrain of BRI-Aβ40/Tg2576 were significantly reduced compared with Tg2576 littermates at all ages (Aβ40 levels at p = 0.01, p < 0.001, and p < 0.001 at 11, 15, and 20 months, respectively, and Aβ42 levels at p < 0.001 at all age). There was no evidence for accumulation of insoluble Aβ in the BRI-Aβ40 mice at any age. B, Because Tg2576 have only minimal accumulation of FA-extractable Aβ up to 15 months of age, comparisons of FA–Aβ levels in BRI-Aβ40 × Tg2576 progeny were determined at 20 months of age. There was an ∼80% reduction in FA–Aβ42 in the hindbrain of bitransgenic BRI-Aβ40/Tg2576 mice compared with single transgenic Tg2576 littermates (p = 0.022 by rank sum test). Decreased FA–Aβ40 levels were also detected but did not reach statistical significance (p = 0.101 by rank sum test). M, Months.

Steady-state RIPA-soluble brain Aβ levels and plasma Aβ levels in BRI-Aβ40/Tg2576 and BRI-Aβ40/BRI-Aβ42A mice before amyloid deposition. To ensure that there was no change in transgene expression levels or alteration in production of Aβ levels in any of the bigenic mice, RIPA-soluble Aβ levels in forebrain, hindbrain, and plasma were analyzed by Aβ sandwich ELISAs. A–C, The levels of RIPA-soluble Aβ40 and Aβ42 in forebrain (A), hindbrain (B), and plasma (C) of BRI-Aβ40/Tg2576 mice were consistent with an additive sum of Aβ levels from their single transgenic littermates at 8 months of age (p > 0.1). D, E, Bitransgenic BRI-Aβ40/BRI-Aβ42A mice had comparable Aβ40 and Aβ42 levels in hindbrain (D) and plasma (E) compared with BRI-Aβ40 and BRI-Aβ42A single transgenic littermates at 2.5 months of age, respectively (p > 0.1). For statistical analysis, see Materials and Methods.

No alteration in amyloidogenic APP processing in BRI-Aβ40/Tg2576 mice. RIPA-soluble forebrain extracts from 8-month-old BRI-Aβ40/Tg2576, Tg2576, BRI-Aβ40, and non-Tg littermates were analyzed by Western blotting. A, B, Blots were probed with 82E1 (anti-Aβ1-16) (A) or CT20 (anti-APP C-terminal 20 amino acids) (B), stripped, and reprobed with anti-β actin to assess loading. C, D, The relative levels of C99 (C) and APP (D) after normalization to β-actin were equivalent between Tg2576 mice and BRI-Aβ40/Tg2576 mice, indicating that there was no alteration in the processing of APP in the bitransgenic mice (p > 0.1). C99 and APP levels were equivalent between non-Tg and BRI-Aβ40 mice that express only endogenous mouse APP (p > 0.1). n = 5 mice per genotype. For statistical analysis, see Materials and Methods.

Aβ modulates premature death. A, Survival rates for progeny of BRI-Aβ40 mice bred with BRI-Aβ42A mice (highest-expressing Aβ42 line) and BRI-Aβ42B mice (lower-expressing Aβ42 line) were calculated using Kaplan–Meier methods. Bitransgenic BRI-Aβ40/BRI-Aβ42A had an accelerated mortality, whereas non-Tg, single transgenic BRI-Aβ40, and BRI-Aβ42A mice did not show any premature death (p < 0.01). BRI-Aβ40/BRI-Aβ42B mice had a decreased premature death rate compared with BRI-Aβ40/BRI-Aβ42A mice (p < 0.01). B, Kaplan–Meier survival curves for progeny of BRI-Aβ40 and BRI-Aβ42A crossed with Tg2576 mice. BRI-Aβ42A/Tg2576 mice had a significantly increased premature death rate compared with Tg2576 littermates (p < 0.05). Although BRI-Aβ40/Tg2576 only had 10% premature death at 7 months of age, Tg2576 mice had ∼30% early death (p < 0.01). There was no difference in survival rates for Tg2576 progeny from breeding with either BRI-Aβ40 mice or BRI-Aβ42A mice (p = 0.884), thus Tg2576 data were pooled. However, only littermates from breeding experiments were used for multiple comparison tests. For statistical analysis, see Materials and Methods.

Aβ40 inhibits Aβ42 aggregation in vitro. Synthetic Aβ40 or Aβ42 peptides were mixed at various molar ratios (1:1.5 and 0.5:0.75 μm). Aβ mixtures were either directly used for analysis (indicated by “−”) or incubated for 2 h at 37°C (indicated by “=”) and electrophoresed on 4–20% Tris-HCl gels under nondenaturing conditions. The blot was probed with Ab9. After a 2 h incubation, the Aβ42-only preparation (Aβ40:Aβ42 ratio; 0:1) formed very high molecular weight (HMW) aggregates (indicated by “⇐” at the top of blot) without any low molecular weight LMW species, whereas the Aβ40 only preparation (Aβ40:Aβ42 ratio; 1:0) remained as a LMW species (indicated by ← at the bottom of blot). When Aβ40 was mixed with Aβ42 (Aβ40:Aβ42 ratio; 0.5:1 and 1:1), Aβ40 inhibited the formation of HMW Aβ42 aggregates, and the vast majority of Aβ42 remained as LMW species (indicated by ← at the bottom of the blot).

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